Transcript Diapositiva 1 - ASCRS/ASOA 2008

COMMON PATTERN IN ELECTROPHORETIC POLYPEPTIDE
PROFILES OF TEAR FLUID OF HEALTHY SUBJECTS AND
CHANGES IN DRY EYE PATIENTS
Leonidas Traipe1, Luis Michea2, Remigio López3.
1 Fundación
Oftalmológica Los Andes
de Medicina, Universidad de Chile
3 ICBM (Biología Celular y Molecular), Facultad de Medicina, Universidad de Chile
2 Facultad
No finantial relationships between the authors and any company or person exist in
regard to the present study
Background

Significant quantitative changes in the production of tear fluid may
occur in Dry-Eye patients (low scores in Schirmer test).

Frequently, those alterations are associated with a lower stability of
the tear film (low Break-up times) what would suggest qualitative
changes in tear macromolecular components.

Direct biochemical analysis of tear is rare quite probably because of
the low abundance of this fluid (5-15 µl/eye; tear flow: 0.5 - 1 µl/min)
Objectives

General: To contribute to the characterization of normal
human tear fluid by the quantitative and qualitative
determination of proteins

Specific: To assess constancy and variability of tear protein
composition among Dry-Eye patients
Methods
Subjects: Forty nine healthy subjects (both sexes, age 21- 60 y.o. (36.4 ± 10.7) who were
not displaying blepharitis, allergy, Dry-Eye or had not experienced ocular surgery were
included. These subjects were no comsumers of medication, alcohol or cigarrettes and
were not contact-lens wearers. Also, fifteen Dry Eye patients (AAO criteria) of identical
age and sexes were included. Some of these patients had been diagnosed with either
rheumatologic or dermatologic diseases. In all cases, informed consents were obtained.
Tear Collection: Absorption by positioning polyurethane minisponges on the outer third of
the margin of the lower lid (see Figure 1). Details in Cornea 25 (3), 312-318 (2006).
Protein Quantification:
Tear aliquots (1-3 microliters) were spotted on cellulose
membranes, fixed and stained with an alcoholic solution of Coomassie blue. After several
washes of the membrane in 7% acetic acid, the dye was eluted in 1 ml of ethanolammonia and the absorbance of the eluate was determined in a spectrophotometer.
Bovine serum albumin was used as standard. Details are given in Cornea 26 (8), 970976 (2007).
Osmolarity. Definite volumes of tear fluid (Vm) were diluted with a definite volume of
double-distilled water (Vw). Osmolarity of both water (Ow) and the diluted tear sample
(Om) were measured in a osmometer. Tear osmolarity (Ot) was calculated by
Ot = (200 x Om) - (Vw x Ow)
Vm
Protein electrophoresis.
One dimensional SDS-PAGE was performed according to the
conventional procedure of Laemmli and bidimensional electrophopresis (NEPHGE)
according to O’Farrrell.
Proteomics. MALDI MS and MALDI MS/MS.
Results
TEAR OSMOLARITY (mOsm/L)
HEALTHY CONTROLS
294 ± 19 (N = 21)
DRY-EYE
332 ± 13 (N = 9) p < 0.001 (Student’s T test)
Tear Fluid Collection
2.
1. Polyurethane minisponges are
positioned for 3 minutes on the outer
third of the lower lid. Eyes can be
either open or closed .
3. Tear fluid is recovered in the
0.5 ml Eppendorf tube by
centrifugation at 2000g x 3 min.
A series of micropipette tips are sectioned
and organized to build up a sort of conical
funnel (left). After tear absorption, the
minisponge is located in the center of the
funnel-like construct which is then
supported on the opening of a 0.5 ml
Eppendorf centrifuge tube (right).
Tear samples in the 1015 µl range
Tear protein quantification
b) After elution in methanol, the dye
solution corresponding to each spot is
read in a spectrophotometer at 600 nm,
and protein concentration is expressed
in relation to the standard protein.
Representative readings
Microliters
a) Cellulose membranes after protein staining.
Aliquots (1-5 µl) of A. Saliva, B. Tear fluid and
C. Standard protein (BSA 1 mg/ml) were
spotted on a cellulose membrane, fixed,
stained with Coomassie blue and washed in
7% acetic acid until clear background.
Representative tear samples (3-µl aliquots) from
4 different subjects
VOLUME (µl)
BSA (1 mg/ml)
TEAR FLUID
3
0.128
0.429
5
0.217
0.768
10
0.430
1.177
Tear protein concentration
around 4 mg/ml
Electrophorertic protein profiles of tear fluid from healthy subjects
SDS-polyacrylamide gel electrophoresis (representative gels)
Lanes were loaded with 30 µg of tear protein per sample. After the fractionation,
proteins were stained with Coomassie blue R-250.
Invariable (79, 18 and 14 kDa) and variable (66 kDa) polypeptides
Normal polypeptide pattern
Some healthy subjects (4%) with a markedly different polypeptide profile (arrow)
Densitometric tracings of electrophoretic polypeptide profiles of
tear fluid from a single healthy subject
A
B
C
D
E
F
Top
Bottom
Both tear collection, electrophoretic fractionation and gel densitometry were
performed in sextuplicate (A through F)
For each tracing, the Y-axis represent band intensities. Tracings of protein migration in gel
fractionation were performed from higher (left) to lower (right) molecular weights.
Band of 66 kDa (arrow in each tracing) corresponds to a highly variable band
Bidimensional NEPHGE fractionation (size and charge) of tear
polypeptides
pH 10
NEPHGE
NEPHGE
pH33
pH
kDa
kDa
Lf
Alb
79
79
66
66
41
41
27
27
24
24
TSPA
Lys
18
18
14
14
Lf, lactoferrin; Alb, Albumin; Lys, lysozyme; TSPA, lipocalin
TEAR POLYPEPTIDE PROFILES OF INDIVIDUAL PATIENTS WITH SEVERE DRY-EYE
LUPUS
Lupus
ROSACEA
SJÖGREN
C
*Tear fluid (6 μl)
from both
eyes were combined.
Loss of major normal bands
Increased intensity of 66 kDa
band.
C, tear sample (7 μl) from a
single healthy subject
Tear samples (7 μl) from the
right and left eyes of a single
subject.
All the normal electrophoretic
bands are clearly more intense
(increased evaporation?)
Tear samples (7 μl)
from the right and
left eyes of a single
subject.
More intense bands
and some degree of
bilateral asymmetry
are observed.
Conclusions

In the normal tear fluid, a general electrophoretic polypeptide profile
consisting of both invariable and variable polypeptides can be
identified.

Lactoferrin, lipocalin and lysozyme are invariable polypeptides
whereas albumin is a highly variable tear component.

Marked alterations in the tear polypeptide composition may occur
among
Dry-Eye patients, such as increased tear protein
concentration as well as selective changes in the presence of
invariable polypeptides